α mem media

OSE cells were isolated from mouse ovaries, maintained, and treated with E2 as previously described [4 (link),52 (link)]. OSE were maintained at 37°C in OSE media that consists of α-MEM media (Corning) with 5% fetal bovine serum, 2ng/mL epidermal growth factor (Sigma), and 0.01mg/mL insulin-transferrin-sodium selenite supplement (Roche). For E2 treatment of OSE, cells were seeded and allowed to normalize to hormone-free media consisting of 5% charcoal-stripped fetal bovine serum in phenol red-free DMEM-F12 media (Sigma) for 48 hours before treating with 100nM E2 (Sigma). An equivalent volume of 100% EtOH (vehicle) was added to control dishes for a final concentration of 0.0002% EtOH. Media was refreshed every 3–4 days and collected for single cell RNA-sequencing or immunofluorescent staining 15 days after E2 treatment. All control and E2-treated OSE cell experiments analyzed in this study are after 15 days in culture.

The human AML cell lines MV4-11, U937, OCI-AML-3 were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). The FLT3-ITD-expressing lines, MOLM-13, MOLM-14, were provided by Dr. Scott Armstrong, Dana Farber Cancer Institute (DFCI), Boston, MA. The wt FLT3-expressing AML line, NB4 (KRAS A18D), was obtained from Dr. Gary Gilliland. MOLM-13, MOLM-14, U937 and FLT3 mutant isogenic BaF3 cells lines were cultured in RPMI 1640 media (Corning, USA) with 10% fetal bovine serum (FBS) and supplemented with 2% L-glutamine 1% penicillin/streptomycin. MV4-11 was cultured in IDMEM media (Corning, USA) with 10% FBS and supplemented with 2% L-glutamine and 1% penicillin/streptomycin. OCI-AML-3 was cultured in α-MEM media (Corning, USA) with 10% FBS and supplemented with 2% L-glutamine and 1% penicillin/streptomycin. All cell lines were maintained in culture media at 37°C with 5% CO₂.